Miguel Lacerda

Immune and Genetic Correlates of Vaccine Protection Against Mucosal Infection by SIV in Monkeys.

A vaccine protecting monkeys against mucosal infection by simian immunodeficiency virus sheds light on immune and genetic correlates of protection. Unraveling Immune Correlates of Vaccine Protection Developing an effective vaccine against HIV-1, the virus that causes AIDS, has been a huge challenge that has stymied AIDS researchers for several decades. A key problem for HIV vaccine trials has been the lack of immune correlates that indicate which antibody and T cell responses in the vaccinees correlate directly with a protective effect. The only HIV vaccine trial to date that has shown a protective effect is the RV144 trial carried out in Thailand between 2003 and 2006, with the final results reported in 2009. In this trial of 16,400 Thai volunteers, those vaccinated with a prime-boost HIV vaccine showed a reduction in the rate of infection by HIV-1 of 31% compared to volunteers given a placebo. The protective effect was seen for up to 3 years after the initial vaccination, but the immune correlates of protection by this vaccine are still not known. In an effort to learn more about possible immune correlates of HIV vaccine protection, Letvin and colleagues used a prime/boost vaccine regimen in monkeys that was similar to that used in the RV144 trial. Monkeys were vaccinated with a plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen and then were challenged with intrarectal doses of one of two isolates of the simian immunodeficiency virus (SIV) every week for 12 weeks. Although the vaccine had no impact on acquisition of the SIVmac251 isolate (which is tough for the monkey immune system to neutralize), the vaccine provided a 50% reduction in infection with the SIVsmE660 isolate (which more readily undergoes neutralization). The authors then examined a variety of immune responses in the protected vaccinated monkeys including cellular, antibody, and innate immune responses; they also examined whether protective host alleles were present in the protected animals. They found that low levels of neutralizing antibodies and a CD4+ T cell response against the HIV envelope (Env) protein correlated with the protective effect. In addition, monkeys that expressed two TRIM5 alleles that help to restrict SIV replication in host cells were protected by the vaccine, whereas monkeys expressing one TRIM5 allele that is permissive for SIV replication were not. This study begins to unravel the immune and genetic correlates of protection in nonhuman primates and highlights the need to scrutinize these types of correlates in future trials of HIV vaccines in human volunteers. The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was about a one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01–negative monkeys challenged with SIVsmE660, no CD8+ T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4+ T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanisms of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.

High prevalence of cisplatin-induced ototoxicity in Cape Town, South Africa

BACKGROUND Cisplatin is administered as the first-line treatment of soft-tissue cancers. It has a reported cure rate of up to 85%, but is associated with a high incidence of ototoxicity, characterised by irreversible bilateral hearing loss and affecting 23 - 50% of adults who receive the drug. OBJECTIVES To determine the incidence of cisplatin-induced ototoxicity at Groote Schuur Hospital (GSH), Cape Town, South Africa. METHODS retrospective cross-sectional study of cisplatin-receiving cancer patients attending GSH between January 2006 and August 2011. RESULTS A total of 377 patients were recorded as receiving cisplatin therapy during the study period. A 300% increase in new cisplatin-receiving patients receiving audiological monitoring was observed between 2006 and 2010. However, only patients with all clinical data as well as baseline and follow-up audiometric analyses were investigated. One hundred and seven such patients were identified, 55.1% of whom developed cisplatin-induced ototoxicity while receiving high-dose (> or = 60 mg/m2) cisplatin treatment. Higher cumulative cisplatin dosages were associated with development of significant hearing loss (p = 0.027). The odds of developing cisplatin-induced hearing loss were elevated for patients with head and neck tumours and lymphoma (p = 0.0465 and p = 0.0563, respectively) and were significantly lower for those with reproductive cancers (p = 0.0371). CONCLUSION Comprehensive audiological monitoring should be available for every patient during cisplatin treatment to minimise the development of disabling hearing loss.

Population Genetics Inference for Longitudinally-Sampled Mutants Under Strong Selection

Longitudinal allele frequency data are becoming increasingly prevalent. Such samples permit statistical inference of the population genetics parameters that influence the fate of mutant variants. To infer these parameters by maximum likelihood, the mutant frequency is often assumed to evolve according to the Wright–Fisher model. For computational reasons, this discrete model is commonly approximated by a diffusion process that requires the assumption that the forces of natural selection and mutation are weak. This assumption is not always appropriate. For example, mutations that impart drug resistance in pathogens may evolve under strong selective pressure. Here, we present an alternative approximation to the mutant-frequency distribution that does not make any assumptions about the magnitude of selection or mutation and is much more computationally efficient than the standard diffusion approximation. Simulation studies are used to compare the performance of our method to that of the Wright–Fisher and Gaussian diffusion approximations. For large populations, our method is found to provide a much better approximation to the mutant-frequency distribution when selection is strong, while all three methods perform comparably when selection is weak. Importantly, maximum-likelihood estimates of the selection coefficient are severely attenuated when selection is strong under the two diffusion models, but not when our method is used. This is further demonstrated with an application to mutant-frequency data from an experimental study of bacteriophage evolution. We therefore recommend our method for estimating the selection coefficient when the effective population size is too large to utilize the discrete Wright–Fisher model.

Identification of broadly neutralizing antibody epitopes in the HIV-1 envelope glycoprotein using evolutionary models

BackgroundIdentification of the epitopes targeted by antibodies that can neutralize diverse HIV-1 strains can provide important clues for the design of a preventative vaccine.MethodsWe have developed a computational approach that can identify key amino acids within the HIV-1 envelope glycoprotein that influence sensitivity to broadly cross-neutralizing antibodies. Given a sequence alignment and neutralization titers for a panel of viruses, the method works by fitting a phylogenetic model that allows the amino acid frequencies at each site to depend on neutralization sensitivities. Sites at which viral evolution influences neutralization sensitivity were identified using Bayes factors (BFs) to compare the fit of this model to that of a null model in which sequences evolved independently of antibody sensitivity. Conformational epitopes were identified with a Metropolis algorithm that searched for a cluster of sites with large Bayes factors on the tertiary structure of the viral envelope.ResultsWe applied our method to ID50 neutralization data generated from seven HIV-1 subtype C serum samples with neutralization breadth that had been tested against a multi-clade panel of 225 pseudoviruses for which envelope sequences were also available. For each sample, between two and four sites were identified that were strongly associated with neutralization sensitivity (2ln(BF) > 6), a subset of which were experimentally confirmed using site-directed mutagenesis.ConclusionsOur results provide strong support for the use of evolutionary models applied to cross-sectional viral neutralization data to identify the epitopes of serum antibodies that confer neutralization breadth.

Anti-Retroviral Therapy Increases the Prevalence of Dyslipidemia in South African HIV-Infected Patients

Purpose Data on the prevalence of dyslipidaemia and associated risk factors in HIV-infected patients from sub-Saharan Africa is sparse. We performed a cross-sectional analysis in a cohort of HIV-infected South African adults. Methods We studied HIV-infected patients who were either antiretroviral therapy (ART)-naive or receiving non-nucleoside reverse transcriptase inhibitor (NNRTI)-based or protease inhibitor (PI)-based ART. Evaluation included fasting lipograms, oral glucose tolerance tests and clinical anthropometry. Dyslipidemia was defined using the NCEP ATPIII guidelines. Results The median age of the participants was 34 years (range 19–68 years) and 78% were women. The prevalence of dyslipidemia in 406 ART-naive and 551 participants on ART was 90.0% and 85%, respectively. Low HDL-cholesterol (HDLC) was the most common abnormality [290/406 (71%) ART-naïve and 237/551 (43%) ART- participants]. Participants on ART had higher triglycerides (TG), total cholesterol (TC), LDL-cholesterol (LDLC) and HDLC than the ART-naïve group. Severe dyslipidaemia, (LDLC> 4.9 mmol/L or TG >5.0 mmol/L) was present in <5% of participants. In multivariate analyses there were complex associations between age, gender, type and duration of ART and body composition and LDLC, HDLC and TG, which differed between ART-naïve and ART-participants. Conclusion Participants on ART had higher TG, TC, LDLC and HDLC than those who were ART-naïve but severe lipid abnormalities requiring evaluation and treatment were uncommon.

Epitope Discovery with Phylogenetic Hidden Markov Models

Existing methods for the prediction of immunologically active T-cell epitopes are based on the amino acid sequence or structure of pathogen proteins. Additional information regarding the locations of epitopes may be acquired by considering the evolution of viruses in hosts with different immune backgrounds. In particular, immune-dependent evolutionary patterns at sites within or near T-cell epitopes can be used to enhance epitope identification. We have developed a mutation–selection model of T-cell epitope evolution that allows the human leukocyte antigen (HLA) genotype of the host to influence the evolutionary process. This is one of the first examples of the incorporation of environmental parameters into a phylogenetic model and has many other potential applications where the selection pressures exerted on an organism can be related directly to environmental factors. We combine this novel evolutionary model with a hidden Markov model to identify contiguous amino acid positions that appear to evolve under immune pressure in the presence of specific host immune alleles and that therefore represent potential epitopes. This phylogenetic hidden Markov model provides a rigorous probabilistic framework that can be combined with sequence or structural information to improve epitope prediction. As a demonstration, we apply the model to a data set of HIV-1 protein-coding sequences and host HLA genotypes.

Recreational fish-finders—an inexpensive alternative to scientific echo-sounders for unravelling the links between marine top predators and their prey

Studies investigating how mobile marine predators respond to their prey are limited due to the challenging nature of the environment. While marine top predators are increasingly easy to study thanks to developments in bio-logging technology, typically there is scant information on the distribution and abundance of their prey, largely due to the specialised nature of acquiring this information. We explore the potential of using single-beam recreational fish-finders (RFF) to quantify relative forage fish abundance and draw inferences of the prey distribution at a fine spatial scale. We compared fish school characteristics as inferred from the RFF with that of a calibrated scientific split-beam echo-sounder (SES) by simultaneously operating both systems from the same vessel in Algoa Bay, South Africa. Customized open-source software was developed to extract fish school information from the echo returns of the RFF. For schools insonified by both systems, there was close correspondence between estimates of mean school depth (R2 = 0.98) and school area (R2 = 0.70). Estimates of relative school density (mean volume backscattering strength; Sv) measured by the RFF were negatively biased through saturation of this system given its smaller dynamic range. A correction factor applied to the RFF-derived density estimates improved the comparability between the two systems. Relative abundance estimates using all schools from both systems were congruent at scales from 0.5 km to 18 km with a strong positive linear trend in model fit estimates with increasing scale. Although absolute estimates of fish abundance cannot be derived from these systems, they are effective at describing prey school characteristics and have good potential for mapping forage fish distribution and relative abundance. Using such relatively inexpensive systems could greatly enhance our understanding of predator-prey interactions.

Effects of CYP2B6 and CYP1A2 Genetic Variation on Nevirapine Plasma Concentration and Pharmacodynamics as Measured by CD4 Cell Count in Zimbabwean HIV-Infected Patients

The extremely high prevalence of HIV/AIDS in sub-Saharan Africa and limitations of current antiretroviral medicines demand new tools to optimize therapy such as pharmacogenomics for person-to-person variations. African populations exhibit greater genetic diversity than other world populations, thus making it difficult to extrapolate findings from one population to another. Nevirapine, an antiretroviral medicine, displays large plasma concentration variability which adversely impacts therapeutic virological response. This study, therefore, aimed to identify sources of variability in nevirapine pharmacokinetics and pharmacodynamics, focusing on genetic variation in CYP2B6 and CYP1A2. Using a cross-sectional study design, 118 HIV-infected adult Zimbabwean patients on nevirapine-containing highly active antiretroviral therapy (HAART) were characterized for three key functional single nucleotide polymorphisms (SNPs), CYP2B6 c.516G>T (rs3745274), CYP2B6 c.983T>C (rs28399499), and CYP1A2 g.-163C>A (rs762551). We investigated whether genotypes at these loci were associated with nevirapine plasma concentration, a therapeutic biomarker, and CD4 cell count, a biomarker of disease progression. CYP2B6 and CYP1A2 were chosen as the candidate genes based on reports in literature, as well as their prominence in the metabolism of efavirenz, a drug in the same class with nevirapine. Nevirapine plasma concentration was determined using LC-MS/MS. The mean nevirapine concentration for CYP2B6 c.516T/T genotype differed significantly from that of 516G/G (p < 0.001) and 516G/T (p < 0.01) genotypes, respectively. There were also significant differences in mean nevirapine concentration between CYP2B6 c.983T > C genotypes (p = 0.04). Importantly, the CYP1A2 g.-163C>A SNP was significantly associated with the pharmacodynamics endpoint, the CD4 cell count (p = 0.012). Variant allele frequencies for the three SNPs observed in this Zimbabwean group were similar to other African population groups but different to observations among Caucasian and Asian populations. We conclude that CYP2B6 c.516G>T and CYP2B6 c.983T>C could be important sources of nevirapine pharmacokinetic variability that could be considered for dosage optimization, while CYP1A2 g.-163C>A seems to be associated with HIV disease progression. These inter- and intra-population pharmacokinetic and pharmacodynamics differences suggest that a single prescribed dosage may not be appropriate for the treatment of disease. Further research into a personalized nevirapine regimen is required.

REPLICATION CAPACITY OF VIRUSES FROM ACUTE INFECTION DRIVES HIV-1 DISEASE PROGRESSION

ABSTRACT The viral genotype has been shown to play an important role in HIV pathogenesis following transmission. However, the viral phenotypic properties that contribute to disease progression remain unclear. Most studies have been limited to the evaluation of Gag function in the context of a recombinant virus backbone. Using this approach, important biological information may be lost, making the evaluation of viruses obtained during acute infection, representing the transmitted virus, a more biologically relevant model. Here, we evaluate the roles of viral infectivity and the replication capacity of viruses from acute infection in disease progression in women who seroconverted in the CAPRISA 004 tenofovir microbicide trial. We show that viral replication capacity, but not viral infectivity, correlates with the set point viral load (Spearman r = 0.346; P = 0.045) and that replication capacity (hazard ratio [HR] = 4.52; P = 0.01) can predict CD4 decline independently of the viral load (HR = 2.9; P = 0.004) or protective HLA alleles (HR = 0.61; P = 0.36). We further demonstrate that Gag-Pro is not the main driver of this association, suggesting that additional properties of the transmitted virus play a role in disease progression. Finally, we find that although viruses from the tenofovir arm were 2-fold less infectious, they replicated at rates similar to those of viruses from the placebo arm. This indicates that the use of tenofovir gel did not select for viral variants with higher replication capacity. Overall, this study supports a strong influence of the replication capacity in acute infection on disease progression, potentially driven by interaction of multiple genes rather than a dominant role of the major structural gene gag. IMPORTANCE HIV disease progression is known to differ between individuals, and defining which fraction of this variation can be attributed to the virus is important both clinically and epidemiologically. In this study, we show that the replication capacity of viruses isolated during acute infection predicts subsequent disease progression and drives CD4 decline independently of the viral load. This provides further support for the hypothesis that the replication capacity of the transmitted virus determines the initial damage to the immune system, setting the pace for later disease progression. However, we did not find evidence that the major structural gene gag drives this correlation, highlighting the importance of other genes in determining disease progression.

Differences in genetic variants in lopinavir disposition among HIV-infected Bantu Africans

INTRODUCTION Variability in lopinavir (LPV) plasma concentration among patients could be due to genetic polymorphisms. This study set to evaluate significance of variants in CYP3A4/5, SLCO1B1 and ABCC2 on LPV plasma concentration among African HIV-positive patients. MATERIALS & METHODS Eighty-six HIV-positive participants on ritonavir (LPV/r) were genetically characterized and LPV plasma concentration determined. RESULTS & DISCUSSION LPV plasma concentrations differed >188-fold (range 0.0206-38.6 µg/ml). Both CYP3A4*22 and SLCO1B1 rs4149056G (c.521C) were not observed in this cohort. CYP3A4*1B, CYP3A5*3, CYP3A5*6 and ABCC2 c.1249G>A which have been associated with LPV plasma concentration, showed no significant association. CONCLUSION These findings highlight the need to include African groups in genomics research to identify variants of pharmacogenomics significance.